title: The Peroxisomal Targeting Signal 1 in sterol carrier protein 2 is autonomous and essential for receptor recognition.
abstract: BACKGROUND
  The majority of peroxisomal matrix proteins destined for translocation into the peroxisomal lumen are recognised via a C-terminal Peroxisomal Target Signal type 1 by the cycling receptor Pex5p. The only structure to date of Pex5p in complex with a cargo protein is that of the C-terminal cargo-binding domain of the receptor with sterol carrier protein 2, a small, model peroxisomal protein. In this study, we have tested the contribution of a second, ancillary receptor-cargo binding site, which was found in addition to the characterised Peroxisomal Target Signal type 1.
  
  
  RESULTS
  To investigate the function of this secondary interface we have mutated two key residues from the ancillary binding site and analyzed the level of binding first by a yeast-two-hybrid assay, followed by quantitative measurement of the binding affinity and kinetics of purified protein components and finally, by in vivo measurements, to determine translocation capability. While a moderate but significant reduction of the interaction was found in binding assays, we were not able to measure any significant defects in vivo.
  
  
  CONCLUSIONS
  Our data therefore suggest that at least in the case of sterol carrier protein 2 the contribution of the second binding site is not essential for peroxisomal import. At this stage, however, we cannot rule out that other cargo proteins may require this ancillary binding site.
authors.affiliation: EMBL-Hamburg, c/o DESY, Notkestrasse 85, 22603 Hamburg, Germany.
authors.forename: Chris P
authors.initials: CP
authors.lastname: Williams
authors.forename: Nicole
authors.initials: N
authors.lastname: Schueller
authors.forename: Colin A
authors.initials: CA
authors.lastname: Thompson
authors.forename: Marlene
authors.initials: M
authors.lastname: van den Berg
authors.forename: Simon D
authors.initials: SD
authors.lastname: Van Haren
authors.forename: Ralf
authors.initials: R
authors.lastname: Erdmann
authors.forename: Charles S
authors.initials: CS
authors.lastname: Bond
authors.forename: Ben
authors.initials: B
authors.lastname: Distel
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Schliebs
authors.forename: Matthias
authors.initials: M
authors.lastname: Wilmanns
authors.forename: Will A
authors.initials: WA
authors.lastname: Stanley
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D000074435:Peroxisome-Targeting Signal 1 Receptor; D020675:Peroxisomes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D021382:Protein Sorting Signals; D021381:Protein Transport; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D020798:Two-Hybrid System Techniques
chemical_list: D002352:Carrier Proteins; D000074435:Peroxisome-Targeting Signal 1 Receptor; D021382:Protein Sorting Signals; D018160:Receptors, Cytoplasmic and Nuclear; C072087:sterol carrier proteins